Introduction

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The major family of receptors for -aminobutyric acid (GABA), the GABA_A receptors, are members of the "gene superfamily" of "Cys-loop" ligand-gated ion channels (Ortells and Lunt, 1995). The GABA_A receptor is a pentameric complex of protein subunits surrounding a central Cl-permeable pore (Bormann et al., 1987). Native GABA_A receptors usually contain , , and  subunits (McKernan and Whiting, 1996) in a 2:2:1 stoichiometry (Chang et al., 1996; Tretter et al., 1997). The existence of six  subunit isoforms enables considerable anatomical and functional diversity of GABA_A receptors (Fritschy and Mohler, 1995; Sieghart, 1995; Nusser at al., 1996). In particular, the  subunit isoform may influence agonist potency (Levitan et al., 1988), agonist efficacy (Ebert et al., 1994), regulation by benzodiazepines (Wafford et al., 1992), and channel kinetics (Tia et al., 1996; Lavoie et al., 1997). Many general anesthetics are allosteric modulators of the GABA_A receptor (Franks and Lieb, 1994; Harris et al., 1995), and mutation of a critical Ser residue within the second transmembrane domain (TM2) of the GABA_A receptor  subunit can ablate or mask the modulation of the receptor by the anesthetic ethers enflurane (Mihic et al., 1997) and isoflurane (Krasowski et al., 1998). Results obtained with the GABA_A receptor 2 S270I (Mihic et al., 1997) and S270H mutants (Krasowski et al., 1998) strongly suggested an important role for  Ser270 in the regulation of GABA_A receptor function by these anesthetics. Because the side chains of Ile and His are both physically larger and Ile is considerably more hydrophobic than is Ser, we sought to determine the effects of other amino acid substitutions at this position. The multiple point mutants were then used to investigate whether size, hydrophilicity, or some other physical parameter of the residue at this position was relevant to the gating behavior and anesthetic modulation of the GABA_A receptor.

	Materials and Methods

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Site-Directed Mutagenesis. To create the mutant series at GABA_A receptor 2 Ser270, mutations were introduced into the cDNA encoding the human GABA_A receptor 2 subunit at bases 890 to 892, with simultaneous loss of a DdeI restriction site. Oligonucleotides, 24 to 30 bases in length, were obtained from Operon Technologies (Alameda, CA) and 5'-phosphorylated using polynucleotide kinase, and used to create mutations using the unique site-elimination method (Deng and Nickoloff, 1992) (USE kit; Pharmacia Biotechnology Inc., Piscataway, NJ), a two-primer method in which a unique SspI site in the expression vector pCIS2 was mutated concurrently to an alternate restriction site (EcoRV or MluI). SspI digestion then was used to select in favor of mutants, and clones were screened for the appearance of the desired mutation by digestion with DdeI. A few additional mutations were created, at Thr268, Leu269, and Ile271 in GABA_A receptor 1, and at Ser265 in GABA_A receptor 1, using a Pfu polymerase/DpnI selection method (QuikChange; Stratagene, La Jolla, CA). All restriction enzymes and polynucleotide kinase were obtained from New England Biolabs (Beverly, MA). The sequences of all cDNA inserts were confirmed throughout by double-stranded sequencing (Sequenase 2.0; U.S. Biochemical Corp., Cleveland, OH), using appropriate oligonucleotide sequencing primers, and are available for inspection on request.

Cell Culture and Transfection. Wild-type or mutant receptor GABA_A receptor cDNAs were expressed via the vector pCIS2, which contains one copy of the strong promoter from cytomegalovirus and a polyadenylation sequence from simian virus 40. These constructs were used to transfect human embryonic kidney (HEK)293 cells (American Type Culture Collection, Rockville, MD), as described previously (Pritchett et al., 1988). HEK293 cells were maintained in culture on glass coverslips; cells were passaged weekly by trypsin treatment up to 15 times before being discarded and replaced with early passage cells. Each coverslip of cells was transfected using the CaPO₄ precipitation technique (Okayama and Chen, 1987). One to five micrograms of each cDNA was used per coverslip; the cDNA was in contact with the cells for 24 h in an atmosphere containing 3% CO₂ before being removed and replaced with fresh culture medium in an atmosphere of 5% CO₂.

Electrophysiology. The coverslips were transferred, between 24 and 72 h after removal of the cDNA, to a large chamber (60 ml) and perfused continuously (20 ml/min) with extracellular medium. Recordings from HEK293 cells were made using the whole-cell patch-clamp technique, as described previously (Koltchine et al., 1996). Patch pipettes contained: 145 mM N-methyl-D-glucamine hydrochloride, 5 mM dipotassium ATP, 1.1 mM EGTA, 2 mM MgCl₂, 5 mM HEPES/KOH, 0.1 mM CaCl₂ (pH 7.2). Pipette resistance was 4 to 5 M. The extracellular medium contained: 145 mM NaCl, 3 mM KCl, 1.5 mM CaCl₂, 1 mM MgCl₂, 6 mM D-glucose, 10 mM HEPES/NaOH (pH 7.4). HEK293 cells were voltage-clamped at 60 mV. In addition to the continuous slow bath perfusion, drugs and solutions were applied rapidly to the cell by local perfusion using a motor-driven solution exchange device (Bio Logic Rapid Solution Changer RSC-100; Molecular Kinetics, Pullman, WA). Laminar flow was achieved by driving all solutions at identical flow rates via a multichannel infusion pump (Stoelting; Wood Dale, IL). Loss of the anesthetic isoflurane using this perfusion device has been measured using gas chromatography and represents only 5 to 10% of the total applied drug concentration (M. D. Krasowski, unpublished observations). The solution changer was driven by protocols in the acquisition program pCLAMP5 (Axon Instruments, Foster City, CA), as described previously (Koltchine et al., 1996). Responses were digitized (TL-1-125 interface; Axon Instruments) using pCLAMP5 (Axon Instruments). Numerical data are presented throughout as mean ± S.E. ATP was obtained from Calbiochem and isoflurane from Ohmeda (Madison, WI); all other chemicals were obtained from Sigma (St. Louis, MO).

Data Analysis, Curve Fitting, and Determination of Parameters of Dose-Response Curves. Control GABA concentration-response data were expressed as a fraction of the maximal response to GABA in each cell, allowing normalized data from different cells to be combined. Pooled data were fitted, using a weighted sum of least-squares method, to a Hill equation of the form:

(1)

where I is the whole-cell current amplitude expressed as a percentage of the current maximal peak current (I_max,) [GABA] is the GABA concentration, EC₅₀ is the GABA concentration eliciting a current equal to half of I_max, and n is the Hill coefficient. Percentage potentiation of a submaximal GABA response by the anesthetic isoflurane was then calculated as the percentage increase above the control (EC₂₀) response to GABA in the presence of anesthetic. Statistical comparisons were made, and significance was assessed using Student's t test.

	Results

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Eleven mutant GABA_A receptors harboring a mutation in the  subunit at Ser270 [2(S270X)1] were studied by whole-cell voltage clamp, after transient expression in HEK-293 cells. First, reproducible whole-cell Cl currents activated by 50 nM to 1 mM GABA were recorded in the absence of other drugs. In all mutant receptors, the maximal response amplitude was between 30 and 100% of that in the wild-type GABA_A receptor (Table 1). Figure 1A shows that in the wild-type receptor, 5 µM GABA recruited a current that was ~30% of the maximal response activated by 500 µM GABA. When Ser270 was mutated to Trp, the same concentration of GABA elicited a near-maximal response; conversely, when Ser270 was mutated to Ala, only 2% of the maximum current was obtained at 5 µM GABA.

View this table: [in this window] [in a new window]   TABLE 1 A summary of the characteristics of GABA responses in the 2(S270X)1 series of GABAA receptors EC50 concentration, Hill coefficient (nH), and Imax are given for each receptor as mean ± S.E. for N cells determined, using the methods described. Statistical significance was assessed using a Student's t test.

View larger version (27K): [in this window] [in a new window]   Fig. 1.   HEK293 cells coexpressing mutant 2(S270X) and wild-type 1 GABAA receptor subunits form functional GABA-activated Cl channels. A-C, representative currents elicited by 2-s applications of 1, 5, 50, and 500 µM GABA recorded from cells expressing receptors made up of 2(S270W)1 (A), wild-type 21 (B), and 2(S270G)1 subunits (C). Notice the modest current activated by 5 µM GABA when 2 Ser270 is mutated to Gly, whereas the same concentration of GABA activates near-maximal current in receptors in which Ser270 is mutated to Trp. The bars above the current traces indicate the period of GABA application and are labeled with the GABA concentration in µM. D, concentration-response curves for GABA in wild-type (S), and 2(S270G)1, 2(S270C)1, and 2(S270W)1 mutant GABAA receptors. E, concentration-response curves for GABA in wild-type (S), and 2(S270A)1, 2(S270H)1, and 2(S270Y)1 mutant GABAA receptors.

Full concentration-response curves for GABA were determined in cells expressing the wild-type GABA_A receptor and the 2(S270X)1 receptor mutants. The currents activated by at least seven concentrations of GABA were expressed as a fraction of the maximal GABA response, and these normalized data were fitted by a Hill equation (see Methods, equation 1). Mutation of Ser270 to smaller amino acid residues (Ala and Gly) produced a rightward shift in the GABA concentration-response curve; conversely, mutating Ser270 to larger amino acid residues such as His and Tyr caused a leftward shift in the curves (Fig. 1B). Concentration-response curves also were constructed for wild-type and mutant 21 and 212s GABA_A receptors (Table 2). As described previously (Draguhn et al., 1990; Smart et al., 1991; Horenstein and Akabas, 1998), the coexpression of the 2s subunit along with the subunits was detected by, and associated with, changes in zinc sensitivity of the resulting GABA_A receptor. The inclusion of the 2s subunit had little or no effect on the GABA EC₅₀ in receptors containing either wild-type or mutant 2 subunits (Fig. 2). Therefore, we suggest that the data from 2(S270X)1 GABA_A receptors also apply to 2(S270X)12 GABA_A receptors. The isoflurane experiments described below were not repeated in the presence of the 2 subunit, because this subunit is not required for modulation by isoflurane (Harrison et al., 1993; Krasowski et al., 1998).

View this table: [in this window] [in a new window]   TABLE 2 A summary of GABA response data for the 2(S270X)12 series of GABAA receptors and for mutant GABAA receptors of the form 2*1, in which mutations are made in residues neighboring Ser270 EC50 concentration, Hill coefficient (nH), and Imax are given for each receptor as mean ± S.E. for N cells determined, using the methods described. Statistical significance was assessed using Student's t test.

View larger version (32K): [in this window] [in a new window]   Fig. 2.   Inclusion of the  subunit does not alter significantly the GABA concentration-response curve in GABAA receptors consisting of 2(S270X)1.

The effects of Trp mutations at positions adjacent to 2 Ser270 and at the 1 homolog Ser265 also were determined. The 2(T268W) mutant expressed poorly or not at all (typically <50 pA GABA current). The 2(I271W) and 2(L269W) mutations had little effect on the GABA concentration-response relationship. Mutation of Ser265 to Trp in the 1 subunit reduced the GABA EC₅₀, but less so than for the 2(S270W) mutant (Table 2). Coexpressing 2(S270W) and 1(S265W) mutant subunits produced a receptor with a similar GABA EC₅₀, but weak expression.

Clinically relevant concentrations of anesthetics such as isoflurane (0.2-0.7 mM) enhance submaximal responses in neuronal GABA_A receptors (Jones et al., 1992). This enhancement is associated with a parallel leftward shift of the GABA concentration-response curve for the wild-type GABA_A receptor (Fig. 3A), consistent with an increase in the apparent affinity of GABA in the presence of the anesthetic. This leftward shift in the GABA concentration-response curve is strikingly similar to that observed on mutagenesis of Ser270 to Trp (Fig. 3B). Potentiation of receptor function by isoflurane was assessed by using an EC₂₀ concentration of GABA appropriate for the receptor under study. Figure 3C shows the modulation by isoflurane of responses to a submaximal (EC₂₀) concentration of GABA in cells expressing wild-type 21, mutant 2(S270A)1, and mutant 2(S270W)1 GABA_A receptors. In the 2(S270A) receptor mutant, the magnitude of potentiation by isoflurane is similar to that seen in the wild-type GABA_A receptor, whereas in the 2(S270Y) mutant, isoflurane does not enhance GABA currents. Table 3 shows the percentage modulation by 0.5 and 1 mM isoflurane of GABA-activated currents in the 11 2(S270X)1 mutant receptors. The largest potentiation was observed in the wild-type; 2(S270A)1 showed identical potentiation. Mutation of Ser270 to Gly, Cys, or Thr resulted in modest anesthetic potentiation; Phe, Ile, His, and larger substitutions rendered the receptor insensitive to clinically relevant concentrations of isoflurane. An extensive study of the concentration-effect curve for GABA potentiation by isoflurane was made in three mutant receptors: 2(S270C)1, 2(S270Y)1, and 2(S270W)1, and compared with the results obtained in the wild-type receptor (Fig. 3D). These data suggest that the effects of Cys and Thr mutations result from a decrease in efficacy for potentiation rather than from a change in the affinity of the receptor for isoflurane. The mutant receptors 2(L269W)1 and 2(I271W)1 showed near-normal potentiation by isoflurane (data not shown).

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Trp mutagenesis at  Ser270 mimics the effect of isoflurane on GABAA receptor function. A, leftward shift of the GABA concentration-response curve for the wild-type 21 GABAA receptor in the presence of 1 mM isoflurane. B, leftward shift of the GABA concentration-response curve for the 2(S270W)1 GABAA receptor relative to the wild-type 21 GABAA receptor. C, allosteric potentiation of submaximal (EC20) GABA responses occurs in the 2(S270X)1 series of GABAA receptors when the 270 substituent is small (Ser, Ala), but not when it is larger (Tyr). D, concentration-response curves for isoflurane potentiation of an EC20 concentration of GABA in wild-type (S), and mutant 2(S270C)1, 2(S270Y)1, and 2(S270W)1 mutant GABAA receptors.

View this table: [in this window] [in a new window]   TABLE 3 A summary of the effects of 0.5 and 1 mM isoflurane on the 2(S270X)1 series of GABAA receptors Percent potentiation of EC20 GABA responses by 0.5 and 1.0 mM isoflurane are given for each receptor as mean ± S.E. for N cells determined, using the methods described. Statistical significance was assessed using a Student's t test.

	Discussion

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Gating of Receptor Mutants by GABA. We conclude from our data that substitution at 2 Ser270 did not compromise ion channel function, consistent with the notion that these point mutations do not induce large changes in secondary structure, at least in the region of the pore. No mutation at Ser270 completely abolished gating by GABA, consistent with the idea that the structure of the agonist binding site also was not disrupted dramatically. The mutations 2(L269W) and 2(I271W) had little effect on receptor function, suggesting again no large changes in secondary structure in this region. These results highlight the significance of the  Ser270 residue. In studies of mutations in the highly homologous and presumably structurally related glycine receptor, it appears that mutations in the nearby TM2 to TM3 linker region alter the transduction mechanism between agonist binding and channel gating, but do not affect ligand binding per se (Lynch et al., 1997).

View larger version (29K): [in this window] [in a new window]   Fig. 4.   The EC50 for GABA within the series of 2(S270X)1 mutant GABAA receptors is correlated inversely with the molecular volume of the amino acid at position 270 on the GABAA-R 2 subunit. The correlation of the physical properties of the amino acid at position 270 with GABA EC50 values obtained from Hill equation fits to GABA concentration-response data (see Materials and Methods, Fig. 3, and Table 1) showed (A) a strong negative correlation with molecular volume (T = 9.25, p < .0005, r = 0.95), whereas no significant correlations were found with (B) side-chain polarity (T = 0.78, p > .22, r = 0.24), (C) hydrophilicity (T = 0.91, p > .18, r = 0.28), or (D) hydropathicity (T = 0.37, p > .36, r = 0.11). Physical parameters are from Harpaz et al. (1994), Hopp and Woods (1981), Kyte and Doolittle (1982), and Zimmerman et al. (1968).

Regulation of Receptor Mutants by Isoflurane. When the GABA_A receptor 2 Ser270 mutants were tested for modulation by isoflurane, we noted that there appeared to be a limiting volume for the side chains, such that amino acid residues with small side chains, such as Ser or Ala, permitted isoflurane modulation, whereas larger side chains such as Cys or Thr allowed only weak modulatory effects of isoflurane (Fig. 5). Many potential explanations exist for the data for the 2 Ser270 mutants and the effects of isoflurane on the wild-type and mutant GABA_A receptors. Without structural information on this part of the receptor molecule, such ideas necessarily must remain speculative. One possibility is that residue 2 Ser270 is involved in structural rearrangements that accompany gating, and that isoflurane, like GABA, binds elsewhere on the receptor molecule to influence the transduction of agonist binding into channel opening. We prefer at present to speculate that the wild-type receptor, with Ser present at 270, contains one or more cavities of finite size within the GABA_A receptor protein, possibly contained entirely within a single  subunit. Mutation of 2 Ser270 to very large residues, such as Phe, Tyr, or Trp (side chain volumes 130, 133, or 168 Å³, respectively; Harpaz et al., 1994), may stabilize the open state of the channel relative to the closed state by occlusion of this cavity. Similarly, the cavity may be filled partially by an isoflurane molecule (molecular volume 140ų; J. Trudell, personal communication), stabilizing the open state in similar manner. The existence, although not the location, of such cavities in ion channel proteins has been inferred from studies of the "cutoff" in activity in the N-alcohol series as chain length increases. Not only do different ligand-gated ion channels show different alcohol cutoffs (Li et al., 1994; Peoples and Weight, 1995; Mihic and Harris, 1996), suggesting cavities of discrete size in each receptor molecule, but the alcohol cutoff actually can be manipulated by mutagenesis within TM2 and TM3 (Wick et al., 1998).

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Potentiation of submaximal GABA responses by isoflurane within the series of 2(S270X)1 mutant GABAA receptors declines with increasing molecular volume of the amino acid at position 270. Each amino acid is represented by the usual three-letter abbreviation. The line represents a best fit to the data but has no theoretical significance.

Conclusion. Whatever the physical mechanisms involved in GABA_A receptor modulation by anesthetics such as isoflurane, 270 certainly influences the behavior of the GABA_A receptor ion channel. Although it is unlikely to participate directly in permeation, given an -helical structure for TM2 (Xu and Akabas, 1996), Ser270 probably lies physically close to the ion channel and may play an important role in channel gating. Furthermore, this residue clearly determines the modulation of the GABA_A receptor by isoflurane and may represent one determinant of an anesthetic binding cavity.